The regulation of the overall renal tubular Pi transport by dietary, hormonal, or metabolic factors occurs via the proximal tubular apical brush border membrane (BBM) sodium gradient-dependent Pi (NaPi) cotransport proteins. Apical entry of Pi is mediated by coordinated actions of NaPi-2a, NaPi-2c, and PiT-2. Chronic alterations in dietary Pi, PTH, FGF23, Klotho, and other metabolic and dietary factors change the abundance of these transporters in BBM. There are however several important differences between these NaPi cotransporters the most important being that a) the time scale kinetics of the regulation of the NaPi transporters by dietary Pi and PTH are quite different, and b) there is also increasing evidence for the differential regulation of the NaPi transporters. This proposal will test the HYPOTHESES that NaPi-2a, NaPi-2c and PiT-2 are localized in distinct brush border membrane microdomains or nanodomains, resulting in: 1) differential regulation of lateral diffusion and clustering of the NaPi transporters in the brush border membrane; 2) differential interactions with PDZ and ERM domain containing proteins, including NHERF-1, PDZK-1, and ezrin; 3) differential rates of recruitment to endocytic pathways and/or differential mechanisms of endocytosis and internalization from the brush border membrane. AIM 1 will determine if NaPi-2a, NaPi-2c and PiT-2 are localized in distinct BBM microdomains or nanodomains resulting in differential regulation of lateral diffusion and clustering of the NaPi transporters in the BBM in response to alterations in PTH, Pi, membrane lipid composition and actin dynamics. AIM 2 will determine if NaPi-2a, NaPi-2c and PiT-2 have differential interactions with PDZ -ERM domain proteins, including NHERF-1, PDZK-1, and ezrin, which result in the differential internalization rate of the NaPi transporters in response to alterations in PTH, Pi and lipids. AIM 3 will determine if NaPi-2a, NaPi-2c and PiT-2 have differential rates of recruitment to endocytic pathways and/or differential mechanisms of endocytosis and internalization from the brush border membrane in response to PTH, Pi and lipids. PUBLIC HEALTH RELEVANCE: Disorders of Pi concentration are common clinical problems in aging, diabetes, cancer, chronic kidney disease, and AIDS by altering renal Pi absorption. Hyperphosphatemia results in vascular calcification and cardiac disease. Hypophosphatemia results in impaired energy production and bone mineralization. Genetic mutations of Pi transporters also cause bone disease, kidney stone formation, and kidney calcification.